Protein kinase C (PKC) is an upstream component of the EGF-stimulated (erbB2) pathway whose activity has been linked to motile behavior of cultured human breast cells and to the acquisition of metastatic behavior. Therefore, identification of PKC substrate proteins would offer potential targets for developing anti-metastasis chemotherapies. Recently, this laboratory successfully applied the Traceable Kinase Method to human breast cells by determining that a- tubulin is a substrate of PKCa that consequently promotes motility. A hypothesis to be tested by both in vitro and intracellular approaches is that phosphorylation of a-tubulin at Ser-165 promotes motility by stimulating microtubule elongation. In addition, the MARCKS protein (previously defined by others to be a PKC substrate) has been found by this laboratory to drive motile behavior of human breast cells, and to depend on phospho-a-tubulin and microtubules for its intracellular trafficking. Planned studies are: Specific Aim (1) to investigate the significance of a-tubulin phosphorylation to polymerization in vitro, microtubule elongation in cultured human breast cells, and morphology of colonies formed in 3D cultures. Specific Aim (2) will explore the reciprocal interactions of MARCKS with microtubules in cells that express fluorescent fusion proteins of mutant constructs of these two PKC substrates. These studies will employ motility assays and live cell imaging (confocal microscopy). For Specific Aim (3), the Traceable Kinase Method will be applied to identify and compare substrates of additional PKC isoforms (d and ?) in human breast cells and tumor cells in order to distinguish shared substrates from those that are isoform-specific. This work will identify new chemotherapeutic targets and disease-related biomarkers for control and detection of metastatic human breast cancer. PUBLIC HEALTH RELEVANCE: This proposal will investigate the protein substrates of protein kinase C (PKC), an enzyme whose activity is critical to the ability of breast cells to spread throughout the body ("metastasis"). The "Traceable Kinase Method" will be used to identify direct protein substrates of PKC, and the biological effects mediated by these proteins will be characterized. This work will identify new chemotherapeutic targets and disease-related biomarkers for control and detection of metastatic human breast cancer.